Retrospective Study of Infections with Corynebacterium diphtheriae Species Complex, French Guiana, 2016–2021

Human infections with Corynebacterium diphtheriae species complex (CdSC) bacteria were rare in French Guiana until 2016, when the number of cases diagnosed increased. We conducted an epidemiologic, multicenter, retrospective study of all human CdSC infections diagnosed in French Guiana during January 1, 2016–December 31, 2021. A total of 64 infectious episodes were observed in 60 patients; 61 infections were caused by C. diphtheriae and 3 by C. ulcerans. Estimated incidence increased from 0.7 cases/100,000 population in 2016 to 7.7 cases/100,000 population in 2021. The mean patient age was 30.4 (+23.7) years, and male-to-female ratio was 1.7:1 (38/22). Of the 61 C. diphtheriae isolates, 5 tested positive for the diphtheria toxin gene, and all results were negative by Elek test; 95% (61/64) of cases were cutaneous, including the C. ulcerans cases. The increase in reported human infections underscores the need to raise awareness among frontline healthcare practitioners to improve prevention.

For each sample, the remaining processed reads were assembled and scaffolded using SPAdes v3.12.0.

Phylogenetic analyses
For the phylogenetic analyses, Panaroo v1.2.3 was used to generate from the assembled genomic sequences, a core genome used to construct a multiple sequence alignment (cg-MSA).
The genome sequences were first annotated using prokka v1.14.5 with default parameters, resulting in GFF files.Protein-coding gene clusters were defined with a threshold of 70% amino acid identity, and core genes were concatenated into a cg-MSA when present in 95% of genomes.IQtree version 2 was used to build a phylogenetic tree based on the cg-MSA, with the best fitting model GTR+F+I+G4.The tree was constructed from 1,903 core genome loci, for a total alignment length of 1,927,340 bp (77% of NCTC13129 genome length, of 2,488,635 bp).

cgMLST and nomenclature of sublineages
The MLST and cgMLST genotypes (cgST) were defined using the Institut Pasteur C. diphtheriae species complex database at https://bigsdb.pasteur.fr/diphtheria.Only for the C. diphtheriae isolates, the nomenclature 'sublineages' (SL; 500 mismatches) and 'genetic clusters' (GC; 25 mismatch threshold) were defined as previously proposed by Guglielmini et al. (5).C. diphtheriae isolates can be classified into 7-gene MLST sequence types and into phylogenetic sublineages, which represent deep phylogenetic subdivisions of the population structure of this species that are highly concordant with ST classifications.GC are much narrower genetic subdivisions of SLs that have been defined as groups of isolates that have, among themselves, genetic distances compatible with outbreaks or recent transmission.For C. ulcerans, we used the cgMLST scheme developed recently and incorporated into the BIGSdb-Pasteur diphtheria platform (https://bigsdb.pasteur.fr/diphtheria/).

Calculation of impact estimates
[(number of CsDS cases diagnosed in the year "n" x 100,000) / number of inhabitants of French Guiana in the year "n"] We took the number of cases per year between 2016 and 2021 described in Figure 3 as the numerator as follows: • 2016: number of cases: 2

Ethical Approval (Details)
In conformity with French legislation, this retrospective study, not involving human persons, adheres to the "Reference Methodology" MR-004, with CH's compliance commitment dated 21/12/2021.A privacy impact analysis was conducted, a study summary is available on the Health-Data-Hub (NN° F20220825152116).Its legal basis is a public interest mission.Data were sourced from routine care patient medical files and anonymised.Regulatory steps were taken to inform patients and allow them to refuse.We tried to reach them by telephone, and then sent a postal letter to non-respondents.Collective information was posted in the care areas known to the people, in their mother tongue, with the support of health mediators.For minors, nonopposition from both legal guardians was required.Deceased participants who hadn't objected during their lifetime were included by default.This approach was carried out in compliance with European regulations (https://www.cnil.fr/fr/reglement-europeen-protection-donnees).

Antimicrobial susceptibility phenotypes and associated genotypes
The isolates were mostly susceptible to all antimicrobials tested (https://docs.google.com/spreadsheets/d/1dK5cCe496bdzQExL4wJFLNY-sbh2hEW4/edit?usp = sharing&ouid = 109112878121272539449&rtpof = true&sd = true).Genomic analysis of antimicrobial resistance genes showed the presence of antimicrobial resistance genes or mutations against four classes of antimicrobial agents (Table S2): sulfonamide (n = 11), tetracycline (n = 7), phenicol (n = 4) and rifamycin (n = 2).Genes sul1 (sulphonamide resistance), and tet(W) and tet(A) (tetracycline resistance) were found and associated with cognate phenotypic resistance.The cmx gene was also found in two isolates, but this agent was not tested phenotypically.For rifamycin, the two phenotypically resistant isolates had a point mutation in the rpoB gene leading to the S442Y amino-acid change.Four isolates were multidrug resistant (acquired resistance to ≥3 classes of drugs), carrying at the same time sul1, tet(W) and cmx.They belonged to three different phylogenetic sublineages, implying genetic transfer of the resistance genes.